The publications and other materials used herein to illustrate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Clinical Chemistry Assays
With clinical chemistry analytes we mean in this context metabolites or other components of serum that are commonly measured in the clinical chemistry practice. Examples of such analytes are as follows: Glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, bilirubin, creatinine, total proteins, iron, magnesium, urea, uric acid, ASAT, ALAT, amylase, LDH, GT, alkaline phosphatase, lipase and creatine kinase. In this context, clinical chemistry analytes do not include analytes, which are measured with bioaffinity binding assay techniques, such as immunometric assay, competitive binding assay, antigen bridging assay or agglutination assay methods (The Immunoassay Handbook, 1994, David Wild, Editor, ISBN 0-333-51179-4).
Most often clinical chemistry analytes are assayed with methods based on photometric detection. The assay protocols include addition of one or several reagents to a sample (e.g. blood, plasma, serum, urine, or other body fluid, diluted or undiluted in an appropriate aqueous or non-aqueous diluent) and incubation at constant condition in one or several occasions after or in-between the reagent additions. Finally, absorption of the assay mixture is measured at one or several wavelengths ranges, followed by calculation of the analyte concentration by aid of standard curves and calibrators. Many of the clinical chemistry assays are based on enzyme-catalyzed reaction where the enzyme acts either as the analyte or as an analyte specific reaction catalyst. As a result of the enzyme catalyzed reaction, a change in the structure of a substrate molecule takes place leading to a change in the absorption properties of the substrate. A change in the photometry reading, at an appropriate wavelength, is proportional to the concentration of the analyte in the sample. In case the analyte acts as a substrate and the enzyme acts as a reagent, the reaction can be run in completion and measured end-point, whereas when the enzyme acts as the analyte, the assay reaction is run under strictly controlled conditions, and measured kinetically. In this context, the term kinetic measurement means that the sample is either measured at certain fixed time point (or time points) or, alternatively, if the kinetics of the reaction is well characterized and modeled mathematically, the reaction can be measured at any precise time point (or time points) followed by calculation of the final enzyme activity (concentration in activity units) using the kinetic equations determined before-hand for this particular application. The assay routines and methodology, which are in common use in the clinical chemistry practice, are well described in textbooks and in the literature (Tiez Textbook of Clinical Chemistry, Ed. C. Burtis and E. Ashwood, W. B. Saunders Company). Another type of clinical chemistry assays is those, which do not include enzyme catalysis, but are of pure chemical basis (non-enzymatic reactions). Such analytes include, for example, assay of bilirubin using the diazoreagent, assay of serum protein using the biuret reaction and assay of albumin using BCG (bromocresol green) reagent (Tiez Textbook of Clinical Chemistry, p. 702). These reactions result in formation of a colorful end-product, the concentration of which is measured by photometry using an appropriate wavelength range. The most typical configuration of a photometry analyzer in the clinical chemistry practice includes a single illumination beam and disposable photometry cuvettes. This means, that any variation in the optical length of the disposable cuvettes or in the optical quality of the cuvette walls causes imprecision in the final assay result. Due to this property of photometry, and on the other hand, due to rather strict precision requirements of clinical chemistry assays, the quality requirements focused on the disposable cuvette are very demanding. For this reason the cuvettes for clinical chemistry assays are and remain expensive. In practice, the cuvette is the major constituent of the cost of the photometric clinical chemistry assays.
Some of the clinical chemistry analytes have also been determined by methods based on fluorescence detection (one-photon excited fluorescence). These assays have been realized by replacing the chromogenic substrate (or reagent) used in photometric methods, with a fluorogenic substrate (or reagent). Consequently, the fluorescence signal obtained from the assay mixture is proportional to the concentration of the analyte. Examples of such assays are, to name some, the assay of glucose using glucose oxidase, peroxidase and Amplex Red™ (fluorogenic substrate, trade mark of Molecular Probes, cat no A-12222) as reagents, and the assay of creatinine using creatininase, creatinase, sarcosine oxidase, peroxidase and Amplex Red as reagents. Another example, is the assay of amylase using fluorophore labeled starch as reagent (or other starch analogue such as amylopectine, or an appropriate synthetic reagent such as α-1,4-oligosaccharide), where the labeled starch is working as substrate for the enzyme providing increase of fluorescence as the amylase enzyme digests the polymer reagent (or oligomer)(Fluorescence Microplate Assays, Molecular Probes, Seventh edition, 2002, p. 51). Another example of fluorometric assay methods is the assay of alkaline phosphatase using a phosphate derivatized fluorescein as fluorogenic substrate (Handbook of fluorescent probes and research products, 9th Ed., p. 420, Molecular Probes, Eugene, Oreg., USA, 2002). This assay is based on increase of fluorescence intensity as the analyte hydrolyses the fluorogenic substrate. The fluorescence based assays are, however, mainly used in research laboratories, whereas their use in the clinical chemistry practice is very limited. In general, fluorescence based methods offer orders of magnitudes higher sensitivity, and broader dynamic range than the methods based on photometry. In research laboratories, these properties give a clear advantage, but in clinical chemistry applications, this has not been found that advantageous because the requirements for sensitivity or dynamic range of the clinical chemistry assays are most often rather modest. Instead, accuracy, precision and the reagent cost are generally considered more important criteria for the clinical chemistry assays. In terms of these criteria, fluorometry has not, until to date, been found to provide clear enough advantages over photometry as a detection technique in the clinical chemistry practice.
Applications of Fluorescence in Bioaffinity Assays
One-photon excited fluorescence has found various applications in the field of bioanalytics. Applications such as immunoassays, DNA-hybridization assays and receptor binding assays using fluorescence as detection method have been introduced during the last three decades. These assays utilize specific bioaffinity reactions in determination of the analyte in a sample. The amount of analyte can be determined by monitoring the fluorescence signal that depends on the amount of the bound analyte. These assays can also be based on monitoring of the change in the fluorescence properties upon a specific binding reaction. This change in fluorescence property can be either a change in fluorescence intensity, a change in emission wavelength, a change in decay time or in fluorescence polarization.
Immunoassays have been used extensively in clinical diagnostics for determination of certain diseases or physiological condition. Immunoassays can be categorized to two different types of assays, competitive and non-competitive assays. In the competitive method, a labeled antigen (secondary biospecific reagent) competes with the analyte in binding to a limited quantity of antibody (primary biospecific reagent). The concentration of the analyte can be determined from the proportion of the labeled antigen bound to the antibody or from the proportion of the free fraction of the labeled antigen. In a non-competitive method (immunometric method) the analyte is bound to an excess amount of binding antibody (primary biospecific reagent). An excess of labeled antibody (secondary biospecific reagent) binds to another site of the analyte. The amount of analyte can be determined on basis of the fraction of the labeled antibody bound to the analyte. Physical separation of the bound and free fractions is normally necessary before detection unless the detection principle is able to distinguish the signal of the bound fraction from the signal of the free fraction. Thus, the assay methods are divided in to separation assays and separation-free assays, often also called as heterogeneous and homogeneous assays. [Miyai K., Principles and Practice of Immunoassay, (ed. Price C. P. and Newman D. J.) Stockton Press, New York 1991, 246 and Hemmila I. A., Applications of Fluorescence in Immunoassays, (ed. Winefordner J. D.) John Wiley & Sons, New York 1991]. Coated tube technology is commonly used for separation assays, and in such cases the free fraction is separated with repeated steps of washing.
One-photon excited fluorometry provides sensitivity and a dynamic range that are higher and wider than that of photometry. However, the sensitivity is sufficient only for a limited number of analytes. Time-resolved fluorescence, chemiluminescence and electrochemiluminescence techniques provide improved sensitivity if compared to conventional steady state (i.e. prompt) fluorescence. These techniques have gained popularity both in research and diagnostics field and are considered among the most sensitive analytical techniques (Hemmilä and Mukkala, Crit. Rev. Clin. Lab. Sci. 2001).
Two-Photon Excitation
In 1931 Maria Göppert-Mayer [Ann. Phys. 9 (1931) 273] postulated that a molecule can simultaneously absorb two photons. This phenomenon remained for a long time without any practical use until the intensive laser light sources became available. Two-photon excitation is created when, by focusing an intensive light source, the density of photons per unit volume and per unit time becomes high enough for two photons to be simultaneously absorbed by the same chromophore. The absorbed energy is the sum of the energies of the two photons. The probability of two-photon excitation is dependent on the 2nd power of the photon density. The absorption of two photons is thus a non-linear process of the second order. The simultaneous absorption of the two photons by one chromophore yields a chromophore in excited state. This excited state is then relaxed by spontaneous emission of a photon with higher energy than the photons of the illumination. In this context the process that includes two-photon excitation and subsequent radiative relaxation is called two-photon excited fluorescence (TPE). TPE has usually similar emission properties to those of one-photon excited fluorescence of the same chromophore [Xu C. and Webb W. W., J. Opt. Soc. Am. B, 13 (1996) 481]. The excitation spectrum, however, is sometimes broadened and/or hypsochromically shifted if compared to one-photon excitation spectrum. The molecules, which are excitable by simultaneous absorption of two photons and generate excited states and fluorescence emission, are in this context called two-photon fluorescent dyes.
One of the key features of two-photon excitation is that excitation takes place only in a clearly restricted 3-dimensional (3D) vicinity of the focal point. The outcome of this feature is high 3D spatial concentration of the generated fluorescence emission. Due to the non-linear nature of excitation, minimal background fluorescence is generated outside the focal volume, i.e. in the surrounding sample medium and in the optical components. Another key feature of two-photon excitation is that illumination and emission takes place in essentially different wavelength ranges. A consequence of this property is that leakage of scattered illumination light in the detection channel of the fluorescence emission can be easily attenuated by using low-pass filters (attenuation of at least 10 orders of magnitude). Since the excitation volume is very small (in the range of femtoliters, i.e. 10-15 liters), two-photon excitation is most suitable for observation of small sample volumes and structures.
Bioanalytical Applications of Two-Photon Excited Fluorescence
One of the early reports relative to analytical applications of two-photon excitation was published by Sepaniak et al. [Anal. Chem. 49 (1977), 1554]. They discussed the possibility of using two-photon fluorescence excitation for HPLC detection. Low background and simplicity of the system were demonstrated. Lakowicz et al. [J. Biomolec. Screening 4 (1999) 355] have reported the use of multi-photon excitation in high throughput screening applications. They have shown that two-photon-induced fluorescence of fluorescein can be reliably measured in high-density multi-well plates.
Most of the bioanalytical applications of two-photon excited fluorescence that are described in the literature relate to two-photon imaging microscopy [Denk W. et al. U.S. Pat. No. 5,034,613, Denk W. et al., Science 248 (1990) 73]. The use of two-photon fluorescence excitation in laser scanning microscopy provides inherent 3D spatial resolution without the use of pinholes, a necessity in confocal microscopy. With a simple optical design two-photon excitation microscopy provides comparable 3D spatial resolution to that of ordinary one-photon excited confocal microscopy. The development has also lead to industrial manufacture of two-photon laser scanning microscope systems. The disadvantage of the two-photon excitation technology is the need of an expensive laser capable of generating intense ultra short pulses with a high repetition frequency.
The recent development of less expensive laser technology is very encouraging in regard to usefulness of two-photon fluorescence excitation technology in routine bioanalytical applications [Hänninen P. et al., Nat. Biotechnol. 18 (2000) 548; Soini J. T. et al. Single Mol. 1 (2000) 203; Soini J T (2002) Crit. Rev. Sci. Instr., WO 98/25143 and WO 99/63344]. According to WO 98/25143 and WO 99/63344, instead of expensive mode-locked lasers a passively Q-switched diode-pumped microchip lasers can be used for two-photon excitation. These lasers are monolithic, small, simple and low in cost. WO 98/25143 and WO 99/63344 describe the use of two-photon excited fluorescence in detection of bioaffinity assay. This bioaffinity assay technique employs microparticles as a bioaffinity binding solid phase to which a primary biospecific reagent is bound. This bioaffinity assay technique utilizes a biospecific secondary reagent that is labeled with a two-photon fluorescent dye. According to the methods described in WO 98/25143 and WO 99/63344, bioaffinity complexes are formed on the surface of microparticles, and the amount of bioaffinity complexes is quantified by measuring two-photon excited fluorescence from individual microparticles. Thus, this assay technique enables separation-free bioaffinity assays in microvolumes.
The labeled secondary bioaffinity reagent binds on the surface of microparticles either via an analyte molecule to form three component bioaffinity complexes (non-competitive, immunometric method) or it binds directly to the primary biospecific reagent to form two component bioaffinity complexes (competitive binding method). The primary and secondary biospecific reagents are biologically active molecules, such as haptens, biologically active ligands, drugs, peptides, polypeptides, proteins, antibodies, or fragments of antibodies, nucleotides, oligonucleotides or nucleic acids. According to WO 98/25143 and WO 99/63344 a laser with high two-photon excitation efficiency is focused into the reaction suspension and two-photon excited fluorescence is measured from single microparticles when they float through the focal volume of the laser beam. Alternatively the microparticles can be trapped for a period of fluorescence detection with an optical trap, which is brought about with a laser beam. The trapping of microparticles to the focal point of the laser beam is based on optical pressure that is generated onto the microparticle by the illuminating laser. According to WO 98/25143 optical trapping increases the duration of the particle within the focal volume of the laser beam and increases the duty cycle of the fluorescence detection. The scheme of the optical lay-out of a typical fluorometric device with two-photon excitation is shown in FIG. 1 (ArcDia™ TPX Plate Reader). The construction of the device may vary depending on the particular use and application. The most important components, which characterize the design, are as follows:                The laser beam is focused by a microscope objective lens with a numerical aperture minimum from 0.4 to 0.7.        For trapping the microparticles a two dimensional pietzo driven scanner is employed, which is capable to stop the scan action momentarily when a microparticle is found in the vicinity of the focal volume.        The near infrared laser is a pulse laser with pulse length shorter than 10 nanoseconds, pulse repetition frequency higher than 10 kHz and average beam power in the sample in the order of 100 mW, with TEM 00 mode polarized beam output. A typical laser is a passively q-switched microchip Nd:YAG or Nd:LBS laser [Danailov M B & al., Appl. Phys. B 73, 1-6 (2001)]. An alternative laser would be a mode locked Yb-doped fiber laser [Grudinin A B & al., Optics Letters (2003), Vol. 28 Issue 17 Page 1522]        The fluorescence signal in the visible range of the light spectrum is detected by photomultiplier tubes CPM by using single photon counting.Current Status of In Vitro Diagnostic Testing        
The major sectors of in vitro diagnostics (IVD) testing are: clinical chemistry assays, bioaffinity assays, hematology and microbiology. From these four sectors the most frequent are clinical chemistry assays, however, the highest sales value and growth rate are the bioaffinity assays. The other sectors, microbiology and hematology, together represent only ⅙ of IVD sales volume but are necessary for a complete primary diagnosis and care.
In typical laboratory practice of small and medium size hospitals or health centers, three types of analyzers are commonly needed: 1) a blood cell counting device for hematology, 2) an automated analyzer for clinical chemistry and 3) an immunoassay analyzer. Microbiological testing can be typically performed with rapid stick tests, by visualization of the microbial cultures or by immunoassay, thus, no dedicated instruments are needed for reading the test result.
As discussed above an in vitro diagnostic laboratory needs today at least three major analyzers for performing the variety of the assays that are most frequently requested. Such a high number of analyzers binds capital, and are cost-effective only when the sample throughput rate of the laboratory is high enough to keep the stand-by time of the analyzer as small as possible. According to the current trend in the industrialized countries, the health care system is under very critical cost effectiveness analysis. The government paid reimbursement for the heath care costs is in a pressure to be reduced. Under this pressure both the clinical laboratories and the manufacturers of analyzers and the assay kits are forced to cut down the overall cost of operations per assay and be able to run the IVD service more effectively. As a consequence, centralization of the IVD routines in large laboratories has been taking place. This trend, however, is in contradiction to the need of intensive care, emergency care and polyclinic duty, where IVD results from single patient samples are required without delay. The trend of centralization is nor applicable in rural areas where the distances from the doctor's office to the central hospital is long. Thus, besides to the trend of centralization there is increasing need for point-of-care (POC) testing and distributed IVD analyzers.
Trend for Centralization of IVD
This trend leads to installation of high capacity IVD analyzer in central laboratories. The collection of patient samples would take place either locally in remote doctor's offices or health care centers or in the reception of the central IVD laboratory. In the former case, the patient samples would be collected and transported from the distant regions by a courier to the central IVD laboratory while the latter requires transportation of the patients. Obviously, the direct cost per test is in minimum when the assays are performed in batch manner in a central laboratory by using high capacity analyzers. However, the health care organizations usually focus their attention to the direct assay cost and are not able to count the indirect cost of IVD in the whole health care system. A lot of money is used for transportation of the samples or the patients to the central laboratories and little value is given to the (i) waiting time of a patient, (ii) multiple patient visits or to (iii) stand-alone-time of the samples in non-controlled conditions. In very few cases the overall cost structure of IVD services and logistics is taken into account when IVD methodology and devices are chosen by the authority. In addition, variable waiting time of individual patient samples in non-controlled conditions (temperature, day light exposure) inevitably causes changes in the composition of the sample leading to inaccurate assay result and diagnosis.
Point-of-Care Assays
Point-of-care (POC) platforms are designed for testing of single samples and are characterized by limited assay portfolio. Typically, POC assays are employing non-standard assay methods, produce qualitative or semi-quantitative results, they are operated without supervision of clinical chemistry experts and without connections to databases. POC tests are typically based on the use of prefabricated disposable assay supports. Some of them are based on immunochromatography technique, which incorporate a chromatographic medium and test zone with immobilized immuno reagent. The detection can take place visually or, for example, by photometric, fluorometric, surface plasmon resonance or electroluminescence techniques. POC testing is cost effective in small-scale use—1 000-10 000 per year. In larger quantities, exceeding 10 000 tests per year, POC tests become less cost effective due to high price of disposable assay component. POC devices are practical only in cases where qualitative results are required instantly for single patient samples.
Distributed IVD
In contrast to the trend of centralization, a clear increase in the need for distributed IVD service and analyzers is found. With distributed IVD we mean IVD practices using compact multipurpose IVD analyzers, which are distributed in locations where the samples are taken from the patients, such as local doctor's offices or remote health centers. The analyzers are connected to the database of a central laboratory trough the telecommunication network. The analyzers are operated and supervised by clinical chemistry experts in a central laboratory, thus the analyzers function like chemical sensors positioned close to the patient.
The technology for distributed IVD is not yet very well developed. There is not yet any ideal and suitable methodology and instrumentation commercially available, which would allow cost effective assays for distributed IVD. This is one of the main reasons for the current trend of IVD centralization in industrialized countries. An ideal analyzer for distributed IVD would perform all of the most frequently requested assays, including both clinical chemistry assays and sensitive immunoassays, with a single detector. In order to allow cost effective assays, the analyzer for distributed IVD should perform all assays separation-free, in other words, without washing steps. Such separation-free assay format would significantly simplify the liquid handling robotics, thus making the instrument smaller and less expensive to manufacture. The physical size of the analyzer should be suitable for table-top use. The analyzer should allow operations in reduced assays volumes. This together with separation-free format (no washing steps) would save reagents and liquid consumables, thus increasing cost-efficiency of the technique. The operation of the analyzer should not require personnel with clinical laboratory competence. The cost of the analyzer should not be higher than for example the cost of a small-scale clinical chemistry analyzer or an immunology analyzer. The device should incorporate dedicated data reduction software for clinical chemistry applications and for quantification of the assays and should allow connection to the data network for supervision by an expert in the central laboratory. This means that the analyzers could be distributed in remote places where the samples are taken, while the clinical chemistry expertise, administration of the assay results, quality control and maintenance would be centralized. There are not many analyzers or technologies commercially available, which fulfill these requirements. Small-scale clinical chemistry analyzers and immunoassay analyzers are normally available as separate units. Some of the commercial clinical chemistry analyzers perform also immunoassays in separation-free format. These immunoassays, however, are usually employing either turbidimetry or nephelometry detection principles (agglutination assays), which do not allow high sensitivity assays but are limited to analytes of relatively high clinical reference concentration.
In order to cope with assays of analytes with lower reference concentration, some manufacturers have recently developed analyzers, which combine clinical chemistry assays with immunoassays using heterogeneous immunoassay principle. An example of such analyzer is that of Adaltis (Adaltis Italia S.p.A, Bologna, Italy), which performs clinical chemistry assay by photometry and immunoassays by chemiluminescence detection principle. Heterogeneous immunoassay principle, however, does not allow separation-free assays and is therefore not ideal for distributed IVD. As a conclusion, the IVD market is looking for new instrumental technology that fulfills the requirements as discussed above.
One of the most important problems with the photometric methodology is the need for expensive test cuvette. If the analyzer is equipped with re-usable flow-cuvettes, the cost of cuvette per assay is not a problem. Re-usable flow-cuvettes, however, are not commonly used today due to their sensitivity to analyte and reagent carry-over and contamination. Thus, the majority of the commercial clinical chemistry analyzers employ disposable photometric cuvettes. Due to the inherent nature of photometric detection, the assay result is critically dependent on the optical quality of the test cuvette and on the length of the optical path. These quality requirements necessitate very highly skilled manufacturing technology, thus making the production of the cuvettes expensive and the corresponding assays prone to inaccuracy due to cuvette-to-cuvette variation. For this reason the cuvettes for clinical chemistry assays are and remain expensive. In fact, the cuvette is the major constituent of the cost of clinical chemistry assays that are based on photometry detection.
An additional problem encountered with the precision of the photometric assays is related to the sample matrix interferences. Blood serum and plasma are the typical samples where the metabolites are assayed. The sample can however contain interfering matrix components such as bilirubin, hemoglobin from hemolyzed erythrocytes or turbidity due to particulate lipids. All these substances can interfere strongly in photometric measurements unless the sample is heavily diluted before the assay.
A third problem with photometric detection relates to the assay volumes. The analytical sensitivity of photometric detection is dependent on the length of the optical path. The longer the optical path the higher dilution factor can be applied. This improves tolerance to interference by matrix components and accuracy of detection. Typical optical length with the commercial analyzers is from 5 to 8 mm. Depending on the geometry of the cuvette, this length leads to an assay volume of 100-300 microliter. Such a cuvette is not optimal with immunoassays because the reagents cost of immunoassays is typically ten times higher than the cost of clinical chemistry reagents. Consequently, clinical chemistry analyzers with photometric detection are relative large in size and require rather large reagent and diluent volumes. This property increases the cost of the immunoassays and the advantage of low assay volumes cannot be exploited.
As summary from the discussion above, we can conclude that the IVD market is still missing a technology and analyzer, which would allow both conventional clinical chemistry and high sensitivity immunoassays, and which would perform all of the most frequently requested assays with separation-free methodology in micro-volumes. In addition, the methodology should minimize or eliminate the need of liquid handling other than dilution and dispensing the sample. The methodology should perform the assays with high precision by using low-cost disposable cuvettes and without the need of prefabricated coated tubes or other expensive assay component. The analyzer would thus combine the functions of two large clinical analyzers, the clinical chemistry analyzer and the immunoanalyzer, and thus to allow cost effective assays in microvolumes.